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A rapid and simplified procedure for purification of a cellulase from Fusarium lini
Author(s) -
Vaidya Madhavi,
Seeta R.,
Mishra Chittra,
Deshpande Vasanti,
Rao Mala
Publication year - 1984
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.260260109
Subject(s) - chemistry , chromatography , isoelectric point , cellulase , enzyme assay , isoelectric focusing , cellulose , size exclusion chromatography , enzyme , biochemistry
Abstract An endo‐β‐1,4‐glucanase (EC 3.2.1.4) was obtained in high yields in purified form a culture filtrate of Fusarium lini by an extremely simple method. The method consists of precipitation of the culture filtrate with ammonium sulphate (290 g/L), followed by chromatography of the precipitated fraction on Biogel P‐150. The purification is based on the unusual property of the enzyme being eluted after cytochrome C, even though it molecular weight is 2.8 × 10 4 (by SDS PAGE). The yield of pure enzyme was 6.8 mg/L culture broth. The homogeneity of the enzyme was established by ultracentrifugation, isoelectric focusing, and electrophoresis in polyacrylamide gels containing SDS. The enzyme was isoelectric at pH 8.3 and contained 2.9% carbohydrate. The K m value for carboxymethyl (CM) cellulose was 11.6 mg/mL. The enzyme showed high viscosity reducing activity towards CM cellulose but very low activity with Walseth cellulose and crystalline celluloses such as Avicel and cotton. The purified enzyme has activity towards xylan. The amino acid analysis showed a predominance of acidic and neutral amino acids and low contents of histidine, arginine, and methionine. One‐half of the cysteine content was 11 residues/mol enzyme, and no free–SH group was detectable.